Senescent cell derived inhibitors of DNA synthesis

ABSTRACT

An expression vector cDNA library derived from senescent cells has been used to isolate cDNA clones that encode inhibitors of DNA synthesis. Such inhibitors play a role in cellular senescence and aging. Antisense nucleic acids reduce the inhibition of DNA synthesis.

FIELD OF THE INVENTION

The present invention is in the field of recombinant DNA technology.This invention is directed to a gene sequence and a protein that effectsthe ability of cells to become senescent. This invention was supportedwith Government funds. The Government has certain rights in thisinvention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 07/970,462, filed on Nov. 2, 1992, now U.S. Pat. No. 5,302,70which is a continuation-in-part of U.S. patent application Ser. No.07/808/523 (filed Dec. 16, 1991) now abandoned, herein incorporated byreference.

BACKGROUND OF THE INVENTION

Normal human diploid cells have a finite potential for proliferativegrowth (Hayflick, L. et al., Exp. Cell Res. 25:585 (1961); Hayflick, L.,Exp. Cell Res. 37:614 (1965)). Indeed, under controlled conditions invitro cultured human cells can maximally proliferate only to about 80cumulative population doublings. The proliferative potential of suchcells has been found to be a function of the number of cumulativepopulation doublings which the cell has undergone (Hayflick, L. et al.,Exp. Cell Res. 25:585 (1961); Hayflick, L. et al., Exp. Cell Res. 37:614(1985)). This potential is also inversely proportional to the in vivoage of the cell donor (Martin, G. M. et al., Lab. Invest. 23:86 (1979);Goldstein, S. et al., Proc. Natl. Acad. Sci. U.S.A.) 64:155 (1969);Schneider, E. L., Proc. Natl. Acad. Sci. (U.S.A.) 73:3584 (1976);LeGuilty, Y. et al., Gereontologia 19:303 (1973)).

Cells that have exhausted their potential for proliferative growth aresaid to have undergone "senescence." Cellular senescence in vitro isexhibited by morphological changes and is accompanied by the failure ofa cell to respond to exogenous growth factors. Cellular senescence,thus, represents a loss of the proliferative potential of the cell.Although a variety of theories have been proposed to explain thephenomenon of cellular senescence in vitro, experimental evidencesuggests that the age-dependent loss of proliferative potential may bethe function of a genetic program (Orgel, L. E., Proc. Natl. Acad. Sci.(U.S.A.) 49:517 (1963); De Mars, R. et al., Human Genet. 16:87 (1972);M. Buchwald, Mutat. Res. 44:401 (1977); Martin, G. M. et al., Amer. J.Pathol. 74:137 (1974); Smith, J. R. et al., Mech. Age. Dev. 13:387(1980); Kirkwood, T. B. L. et al., Theor. Biol. 53:481 (1975).

Cell fusion studies with human fibroblasts in vitro have demonstratedthat the quiescent phenotype of cellular senescence is dominant over theproliferative phenotype (Pereira-Smith, O. M. et al., Somat. Cell Genet.8:731 (1982); Norwood, T. H. et al., Proc. Natl Acad. Sci. (U.S.A.)71:223 (1974); Stein, G. H. et al., Exp. Cell Res. 130:155 (1979)).

Insight into the phenomenon of senescence has been gained from studiesin which senescent and young (i.e. non-senescent) cells have been fusedto form heterodikaryons. In order to induce senescence in the "young"nucleus of the heterodikaryon (as determined by an inhibition in thesynthesis of DNA), protein synthesis must occur in the senescent cellprior to fusion (Burmer, G. C. et al., J. Cell. Biol. 94:187 (1982);Drescher-Lincoln, C. K. et al., Exp. Cell Res. 144:455 (1983); Burner,G. C. et al., Exp. Cell. Res. 145:708 (1983); Drescher-Lincoln, C. K. etal., Exp. Cell Res. 153:208 (1984).

Likewise, microinjection of senescent fibroblast mRNA into youngfibroblasts has been found to inhibit both the ability of the youngcells to synthesize DNA (Lumpkin, C. K. et al., Science 232:393 (1986))and the ability of the cells to enter into the S (stationary) phase ofthe cell cycle (Lumpkin, C. K. et al., Exp. Cell Res. 160:544 (1985)).Researchers have identified unique mRNAs that are amplified in senescentcells in viro (West, M. D. et al., Exp. Cell Res. 184:138 (1989);Giordano, T. et al., Exp. Cell Res. 185:399 (1989)).

The human diploid endothelial cell presents an alternative cell type forthe study of cellular senescence because such cells mimic cellularsenescence in vitro (Maciag, T. et al., J. Cell. Biol. 91:420 (1981);Gordon, P. B. et al., In Vitro 19:661 (1983); Johnson, A. et al., MechAge. Dev. 18:1 (1982); Thornton, S. C. et al., Science 222:623 (1983);Van Hinsbergh, V. W. M. et al., Eur. J. Cell Biol. 42:101 (1986);Nichols, W. W. et al., J. Cell. Physiol. 132:453 (1987)).

In addition, the human endothelial cell is capable of expressing avariety of functional and reversible phenotypes. The endothelial cellexhibits several quiescent and non-terminal differentiation phenotypes(Folkman, J. et al., Nature 288:551 (1980); Maciag, T. et al., J. CellBiol. 94:511 (1982); Madri. J. A. et al., J. Cell Biol. 97:153 (1983);Montesano, R., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J.Cell Physiol. 34:460 (1988)).

It has been suggested that the pathway of human cell differentiation invitro involves the induction of cellular quiescence mediated bycytokines that inhibit growth factor-induced endothelial cellproliferation in vitro (Jay, M. et al., Science 228:882 (1985); Madri,J. A. et al., In Vitro 23:387 (1987); Kubota, Y. et al., J. Cell Biol.107:1589 (1988); Ingber, D. E. et al., J. Cell Biol. 107:317 (1989)).

Inhibitors of endothelial cell proliferation also function as regulatorsof immediate-early transcriptional events induced during the endothelialcell differentiation in vitro, which involves formation of thecapillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp.Adv. Oncol. (De Vita, V. T. et al., eds. , J.B. Lippincott.Philadelphia, 42 (1990); Goldgaber, D. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86:7606 (1990); Hla, T. et al., Biochem Biophys. Res. Commun.167:637 (1990)). The inhibitors of cell proliferation that include: 1.Interleukin-la (IL-1a) (Montesano, R. et al., J. Cell Biol. 99:1706(1984); Montesano, R. et al., J. Cell Physiol. 122:424 (1985); Maciag,T. et al. (Science 249:1570-1574 (1990));

2. Tumor necrosis factor (Frater-Schroder, M. et al., Proc. Natl. Acad.Sci. (U.S.A.) 84:5277 (1987); Sato, N. et al., J. Natl. Cancer Inst.76:1113 (1986); Pber, J. P., Amer. J. Pathol. 133:426 (1988); Shimada,Y. et al., J. Cell Physiol. 142:31 (1990));

3. Transforming growth factor-β (Baird, A. et al., Biochem. Biophys.Res. Commun. 138:476 (1986); Mullew, G. et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:5600 (1987); Mairi, J. A. et al., J. Cell Biol. 106:1375(1988));

4. Gamma-interferon (Friesel, R. et al., J. Cell Biol. 104:689 (1987);Tsuruoka, N. et al., Biochem. Biophys. Res. Commun. 155:429 (1988)) and

5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. etal., Cell 42:469 (1985); Doctrow, S. R. et al., J. Cell Biol. 104:679(1987); Montesano, R. et al, J. Cell. Physiol. 130:284 (1987); Hoshi, H.et al., FASAB J. 2:2797 (1988)).

The prospect of reversing senescence and restoring the proliferativepotential of cells has implications in many fields of endeavor. Many ofthe diseases of old age are associated with the loss of this potential.Also the tragic disease, progeria, which is characterized by acceleratedaging is associated with the loss of proliferative potential of cells.Restoration of this ability would have far-reaching implications for thetreatment of this disease, of other age-related disorders, and, of agingper se.

In addition, the restoration of proliferative potential of culturedcells has uses in medicine and in the pharmaceutical industry. Theability to immortalize nontransformed cells can be used to generate anendless supply of certain tissues and also of cellular products.

The significance of cellular senescence has accordingly been appreciatedfor several years (Smith, J. R., Cellular Ageing, In: Monographs inDevelopment Biology; Sauer, H. W. (Ed.), S. Karger, New York, N.Y.17:193-208 (1984); Smith, J. R. et al., Exper. Gerontol. 24:377-381(1989), herein incorporated by reference). Researchers have attempted toclone genes relevant to cellular senescence. A correlation between theexistence of an inhibitor of DNA synthesis and the phenomenon ofcellular senescence has been recognized (Spiering, A.I. et al., Exper.Cell Res. 179:159-167 (1988); Pereira-Smith, O. M. et al., Exper. CellRes. 160:297-306 (1985); Drescher-Lincoln, C. K. et al., Exper. CellRes. 153:208-217 (1984); Drescher-Lincoln, C. K. et al., Exper. CellRes. 144:455-462 (1983)). Moreover, the relative abundance of certainsenescence-associated RNA molecules has been identified (Lumpkin, C. K.et al., Science 232:393-395 (1986)).

Several laboratories have used the "subtraction-differential" screeningmethod to identify cDNA molecules derived from RNA species that arepreferentially present in senescent cells (Kleinsek, D. A., Age 12:55-60(1989); Giordano, T. et al., Exper. Cell. Res. 185:399-406 (1989);Sierra, F. et al., Molec. Cell. Biol. 9:5610-5616 (1989); Pereira-Smith,O. M. et al., J Cell. Biochem. (Suppl 0 (12 part A)) 193 (1988);Kleinsek, D. A., Smith, J. R., Age 10:125 (1987)).

In one method, termed "subtraction-differential" screening, a pool ofcDNA molecules is created from senescent cells, and then hybridized tocDNA or RNA of growing cells in order to "subtract out" those cDNAmolecules that are complementary to nucleic acid molecules present ingrowing cells. Although useful, for certain purposes, the"subtraction-differential" method suffers from the fact that it is notpossible to determine whether a senescence-associated cDNA molecule isassociated with the cause of senescence, or is produced as a result ofsenescence. Indeed, many of the sequences identified in this manner havebeen found to encode proteins of the extra-cellular matrix. Changes inthe expression of such proteins would be unlikely to cause senescence.

SUMMARY OF THE INVENTION

The present invention concerns, in part, the observation that normalhuman cells exhibit a limited replicative potential in vitro and becomesenescent after a certain number of divisions. As the cells becomesenescent, they show several morphological and biochemical .changes,such as enlargement of cell size, changes of extracellular matrixcomponents, unresponsiveness to mitogen stimulation and failure toexpress growth regulated genes.

The present invention identifies an inhibitor of DNA synthesis that isproduced in senescent cells. This inhibitor plays a crucial role in theexpression of the senescent phenotype. The gene coding for the inhibitorwas identified by incorporating a senescent cell cDNA library into amammalian expression vector. The cDNA library was then transfected intoyoung, cycling cells to identify those library members that suppressedthe initiation of DNA synthesis.

Efficient DEAE dextran-mediated transfection enabled the isolation ofputative senescent cell derived inhibitor (SDI) sequences in threedistinct cDNA clones. The expression of one (SDI-1) increased 20 fold atcellular senescence, whereas that of the others (SDI-2 and SDI-3)remained constant.

In summary, the present invention achieves the cloning of an inhibitorof DNA synthesis using a functional assay. This method may be applied toclone other genes involved in negative regulation of the cell cycle,such as tissue specific differentiation and tumor suppression genes.Using this method, three inhibitor sequences have been cloned. One ofthese sequences (SDI-1) appears to be closely related to cellularsenescence.

In detail, the invention provides a nucleic acid molecule that encodes aprotein capable of inhibiting DNA synthesis in a recipient cell.

The invention particularly concerns the embodiment wherein the nucleicacid molecule is DNA, and is incorporated into a DNA plasmid (such aspcDSRαΔ).

The invention also concerns the embodiments wherein the above statednucleic acid molecule is SDI-1, and wherein it has the sequence shown inFIGS. 5A-D <SEQ ID 1>.

The invention also includes the embodiment wherein the nucleic acidmolecule is RNA.

The invention also concerns a nucleic acid molecule (either DNA or RNA)having a sequence complementary to such RNA molecule, and a lengthsufficient to permit the molecules to hybridize to one another underphysiological conditions.

The invention also provides a method for inhibiting DNA synthesis in ahuman cell which comprises providing to the cell an effective amount ofthe above-stated nucleic acid molecule that encodes a protein capable ofinhibiting DNA synthesis in a recipient cell (and especially wherein thecell is a tumor cell, or a cell in in vitro culture.

The invention also provides a method for derepressing an inhibition ofDNA synthesis in a quiescent or senescent human cell which comprisesproviding to the cell an effective amount of a nucleic acid molecule(either DNA or RNA) having a sequence complementary to an RNA moleculethat encodes a protein capable of inhibiting DNA synthesis in arecipient cell, and having a length sufficient to permit the moleculesto hybridize to one another under physiological conditions. Especiallycontemplated is the embodiment wherein the cell is a skin cell or a cellpresent in wound or burn tissue. The invention further contemplates theuse of the agents of the present invention in tissue other than skin,such as lymphocytes, vascular tissue (such as arteries, arterioles,capillaries, veins, etc.), liver, kidney, heart and other muscle, bone,spleen, etc.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of the cDNA cloning and expression vector,pcDSRαΔ (B represents BamHI site).

FIGS. 2A-C identify cDNA clones inhibitory to young cell DNA synthesis.The three different bars represent independent transfectionexperiments, * indicates not done, a negative number indicates labellingindices higher than the controls.

FIG. 3 shows antisense SDI cDNA transfection. Antisense cDNA expressionplasmids were made and co-transfected with pCMVβ into young cells. Lane1: control pcdSRαΔ, lane 2: pcDSRαΔ-SDI-1, lane 3: pcDSRαΔ antiSDI-1,lane 4: pcDSRαΔ-SDI-2, lane 5: pcDSRαΔ-antiSDI-2.

FIG. 4 shows the changes in poly A+ RNA recovery from total RNA duringcellular aging.

FIGS. 5A-5D provides the nucleotide and amino acid sequences of SDI-1cDNA.

DETAILED DESCRIPTION OF THE INVENTION I. Cellular Senescence

Replicative senescence of normal human diploid fibroblasts in culture isa well established and widely accepted model for cellular aging(Hayflick, L., Exp. Cell Res. 37:611-636 (1965); Norwood, T. H., andSmith, J. R., In: Handbook of the Biology of Aging (2nd ed.) C. E. Finchand E. L. Schneider, eds. Van Nostrand, New York pp. 291-311 (1985);Goldstein, S., Science 249:1129-1133 (1990)). After a limited number ofpopulation doublings, as cells become senescent, they lose thecapability to divide and display a large and flattened morphology. Thecausative mechanisms underlying this phenomenon are not yet understood,despite the many observations that characterize senescent cells at thebiochemical and molecular levels.

One- and two-dimensional protein gel analyses have revealed that thereare few senescent cell-specific marker proteins (Lincoln, D. W. et al.,Exp. Cell Res. 154:136-146 (1984); Wang, E., J. Cell Biol. 100:545-551(1985); Scottie, J. et al., J. Cell Physiol. 131:210-217 (1987);Bayreuther, K. et al., Proc. Natl. Acad. Sci. USA. 85:5112-5116 (1988)).Antigenic determinants that specify senescent cells have been found onthe plasma membrane (Porter, M. B. et al., J. Cell Physiol. 142:425-433(1990)). Components of extracellular matrix, such as fibronectin andcollagenase, have been found to be overexpressed in senescent cells(West, M. D. et al., Exp. Cell Res. 184:138-147 (1989); Kumazaki, T. etal., Exp. Cell Res. 195:13-19 (1991)). However, the relevance of theseobservations to cellular senescence is not clear.

Recently, changes in the expression of several growth regulated geneshave been identified. Expression of c-fos cdc2, cyclin A and B have beenfound to be impaired in senescent cells (Seshadri, T., and Campisi, J.,Science 247:205-209 (1990)). Similarly, senescent cells evidence aninability to phosphorylate the retinoblastoma protein (Stein, G. H. etal., Science 249:666-669 (1990)). These observations could potentiallyexplain the inability of the cells to enter S phase, since they are alldeteriorative changes of growth promoting gene expression, however, itis not clear whether they are the cause or result of senescence.

One additional change in gene expression that could have a causal rolein senescence is the inhibitor(s) of DNA synthesis produced by senescentbut not young fibroblasts (see, Spiering, A. I. et al., Exper. Cell Res.195:541-545 (1991). Evidence for the existence of the inhibitor(s) wasfirst obtained from heterokaryon experiments in which senescent cellsinhibited initiation of DNA synthesis in young nuclei within theheterokaryon (Norwood, T. H., et al., Proc. Natl. Acad. Sci. USA.71:2231-2234 (1974); Pereira-Smith, O. M., and Smith, J. R., Somat. CellGenet. 3:731-742 (1982)). Studies with cybrids involving senescentcytoplasts and whole young cells lent further support for the presenceof a surface membrane associated protein inhibitor of DNA synthesis insenescent cells (Dresher-Lincoln, C. K., and Smith, J. R., Exp. CellRes., 153:208-217 (1984)). This was directly demonstrated when surfacemembrane enriched preparations from senescent cells or proteinsextracted from the membranes were found to inhibit DNA synthesis whenadded to the culture medium of young cells (Pereira-Smith, O. M. et al.,Exp. Cell Res. 160:297-306 (1985); Stein, G. H., and Atkins, L., Proc.Natl. Acad, Sci. U.S.A. 83:9030-9034 (1986)). Purification of thatinhibitor by biochemical methods has been unsuccessful to date. However,in microinjection experiments, the presence of a high abundance of DNAsynthesis inhibitory messenger RNA has been demonstrated (Lumpkin, C. K.et al., Science 232:393-395 (1986)).

In order to attempt to clone the gene(s) coding for the DNA synthesisinhibitor(s), a functional screening procedure was employed. This methodled to the isolation and identification of three cDNA species thatexhibit DNA synthesis inhibitory activity when introduced into youngcycling cells. These molecules are referred to herein as "senescent cellderived inhibitors" ("SDI").

II. The Cloning of Inhibitors of Cellular Senescence

In the practice of the present invention, an efficient method for themolecular cloning of the DNA synthesis inhibitory sequences present insenescent human diploid fibroblasts is preferably employed. As is oftenthe case when attempting to clone biologically important genes, it maynot be possible to purify a desired gene responsible for cellularsenescence, even though the activity of its products could be readilydetected.

One method that might be envisioned for identifying such a gene sequencewould be to employ a differential or subtractive screening of asenescent cell derived cDNA library. This method has been used toidentify cDNA molecules that are overexpressed in cells from WernerSyndrome patients (Murano, S. et al., Molec. Cell. Biol. 2:3905-3914(August 1991)). Werner Syndrome is a rare inherited disorder. It ischaracterized by premature aging. The relevance of Werner Syndrome tonatural aging is unknown.

Unfortunately, such screenings would identify a number of genes that,although important for the characterization of senescent cells, wouldnot be primarily responsible for senescence. Furthermore, technicallimitations in cloning full-length cDNA make it difficult to determinethe function of genes cloned by these methods. For these reasons, suchdifferential methods are nether generally suitable, or the mostdesirable method of identifying senescence-related gene sequences.

In contrast, expression screening provides a preferred method foridentifying and isolating such senescence-related gene sequences. Insuch a screening method, the cDNA is cloned directly into a vector thatis capable of expressing the cloned gene in a recipient cell. Therecipient cells can thus be directly screened for any inhibition in DNAsynthesis.

In expression screening, the most important step is the synthesis ofcDNAs. Enzymes should be carefully chosen to be free of impurities. ThecDNA synthesis is preferably repeated several times to ensure thatsatisfactory results (i.e. faithful reverse transcription, and fulllength transcript size) will be obtained. Finally, the cDNA products arepreferably size fractionated to eliminate fragmented and prematurelyterminated cDNA products. Double stranded cDNA products are thenpreferably divided into fractions based on size, i.e., 0.5-2.0, 2.0-4.5,and 4.5-10 kb fractions. The 2-4.5 kb cDNA fraction was used to make thecDNA library on the assumption that many membrane associated proteinshave a relatively high molecular weight. The cDNAs are inserted into asuitable expression vector, preferably pcDSRαΔ, in which the insertedsequences can be transcribed at high levels in young cells.

The most preferred transfection procedure is DEAE dextran-mediatedtransfection, carried out under conditions that allowed for transientexpression in a high percentage of young cycling cells. Since thetransfection frequencies could vary from experiment to experiment, thecDNA pool plasmids were transfected along with a marker plasmid, such aspCMVβ (encoding β-galactosidase), and the labelling index was assayed inonly β-galactosidase positive cells. Generally, co-expression oftransfected genes is quite high, since transfection competent cells willaccept multiple plasmids. This simple co-transfection method enabled theevaluation of DNA synthesis in cells expressing exogenous DNA.

The amount of plasmid to be co-transfected was determined from pilotexperiments. When the correlation between the transfection frequency andthe amount of plasmid added is examined using a marker plasmid, maximumefficiency is obtained at a range of 100-500 ng of plasmid. Taking intoaccount this result, the cDNA library is preferably divided into smallpools in which every pool contained five independent plasmid clones.Then the co-transfection is carried out with approximately 100 ng ofpCMVβ and approximately 400 ng of cDNA plasmid. These parameters werefound to maximize the co-expression of cDNA in β-galactosidase positivecells without decreasing the transfection frequency of the markerplasmid.

After the second round of screening, single plasmids which showed stronginhibition of DNA synthesis can be successfully isolated from the poolthat tested positive during the first round screenings (FIGS. 2A-C). InFIGS. 2A-C, cDNA pools which showed positive in the first roundscreenings were divided into individual plasmid, and transfected again.For every cDNA pool (A, B and C), plasmid No. 1 to 5 represents theresult of each single plasmid transfection. In pool B, No. 1 plasmid wasfound to be only the empty vector. The inhibitory activities of theplasmids are preferably further confirmed by nuclear microinjectionexperiments. Such experiments provide more direct evidence that theisolated plasmids contain sequences capable of inhibiting DNA synthesis.

III. The Molecules of the Present Invention and Their Uses

The present invention contemplates the use of any of a variety ofchemical agents to either inhibit or enable DNA synthesis. Such agentsmay be: (1) an oligonucleotide, (2) a nucleic acid binding protein, or(3) a compound whose structure mimics that of either an oligonucleotideor a nucleic acid binding molecule (i.e. a "peptidomimetic" agent).

The agents of the present invention are capable of either inducing theinhibition of DNA synthesis in active cells, or suppressing suchinhibition in senescent or quiescent cells, they may be used for a widerange of therapies and applications.

Thus, in one embodiment, the present invention provides a means ofisolating cDNA molecules, in functional (i.e. expressible) form, thatare capable of inhibiting DNA synthesis in recipient cells. Such "SDI"nucleic acid molecules, as well as the proteins they encode, and theirpeptidomimetic analogs, have use in inducing a senescent or quiescentstate in a recipient cell. Such induction is desirable in the treatmentof progeria (Badame, A. J., Arch. Dermatol. 125:540 (1989); Hamer, L. etal., Orthoped. 11:763 (1988); Martin, G. M., Natl. Canc. Inst. Monogr.60:241 (1982)); age-related disorders (Martin, G. M., Genome 31:390(1989); Roe, D. A., Clin. Geriatr. Med. 6:319 (1990); Mooradian, A. D.,J. Amer. Geriat. Soc. 36:831 (1988); Alpert, J. S., Amer. J. Cardiol.65:23j (1990)); Alzheimer's disease (Terry, R. D., Monogr. Pathol. 32:41(1990); Costall, B. et al., Pharmacopsychiatry 23:85 (1990)); astheniaand cachexia (Verdery, R. B., Geriatrics 45:26 (1990)), or diseases orconditions in which rapid cellular proliferation is undesirable. In thisrespect, the agents of the present invention can be used therapeuticallyto suppress the rapid proliferation of tumor or tumorigenic cells. Thus,the present invention provide a therapy for treating cancer.

The sequence of the SDI nucleic acid molecules permits one to ascribeand identify protein molecules that can be used to suppress theinhibition of DNA synthesis associated with quiescence and senescence.The amino acid sequence of such molecules can be readily derived fromthe known relationship between the nucleotide sequence of a nucleic acidmolecule, and the amino acid sequence of the protein it encodes. Thepresent invention includes the protein and polypeptide molecules thatwould be synthesisized through the transcription and translation of thedisclosed SDI nucleic acid molecules.

An additional class of molecules that is contemplated by the presentinvention comprises proteins or other molecules (i.e. petidomimeticanalogs) that mimic the function of the proteins expressed from the SDIsequences.

These and other analogs can be readily identified by, for example,exploiting the capacity of the agents of the present invention to induceor to derepress DNA synthesis may be used to identify agents capable ofreversing these processes. Thus, for example, one may incubate cells inthe presence of both an SDI oligonucleotide and a suspected antagonistcompound. The cells would be monitored in order to determine whether thecompound is able to impair the ability of the SDI oligonucleotide toinhibit DNA synthesis. Thus, the present invention includes a "screeningassay" capable of identifying antagonists of the SDI oligonucleotides.Conversely, one may incubate cells in the presence of both an SDIantisense oligonucleotide and a suspected antagonist compound. The cellswould be monitored in order to determine whether the compound is able toimpair the ability of the antisense oligonucleotide to derepress DNAsynthesis. Thus, the present invention includes a "screening assay"capable of identifying antagonists of the antisense oligonucleotides. Ina similar manner, agonists of these agents may alternatively beidentified.

Among the agonist compounds which could be identified through the use ofsuch a screening assay are compounds which could be used to induceinfertility. Similarly, the assay will permit the identification ofcompounds capable of either suppressing or inducing tissue regenerationor vascularization. Such compounds may be useful in the treatment ofcancer.

In addition to their use in expressing proteins and polypeptides, and indefining desirable analogs, the SDI nucleic acid molecules of thepresent invention can be used to produce antisense nucleic acidmolecules capable of binding to an SDI nucleic acid molecule andinhibiting its activity, etc. A particularly preferred such agent isantisense oligonucleotide.

In general, an "antisense oligonucleotide" is a nucleic acid (either DNAor RNA) whose sequence is complementary to the sequence of a target mRNAmolecule (or its corresponding gene) such that it is capable of bindingto, or hybridizing with, the mRNA molecule (or the gene), and therebyimpairing (i.e. attenuating or preventing) the translation of the mRNAmolecule into a gene product. To act as an antisense oligonucleotide,the nucleic acid molecule must be capable of binding to or hybridizingwith that portion of target mRNA molecule (or gene) which mediates thetranslation of the target mRNA. Antisense oligonucleotides are disclosedin European Patent Application Publication Nos. 263,740; 335,451; and329,882, and in PCT Publication No. WO90/00624, all of which referencesare incorporated herein by reference.

The present invention is particularly concerned with those antisenseoligonucleotides which are capable of binding to or hybridizing withmRNA or cDNA molecules that encode an SDI gene product.

Thus, in one embodiment of this invention, an antisense oligonucleotidethat is designed to specifically block translation of an SDI mRNAtranscript can be used to de-repress the inhibition of DNA synthesis ina recipient senescent cell.

One manner in which an anti-SDI antisense oligonucleotide may achievethese goals is by having a sequence complementary to that of thetranslation initiation region of an SDI mRNA and of sufficient length tobe able to hybridize to the mRNA transcript of an SDI gene. The size ofsuch an oligomer can be any length that is effective for this purpose.Preferably, the antisense oligonucleotide will be about 10-30nucleotides in length, most preferably, about 15-24 nucleotides inlength.

Alternatively, one may use antisense oligonucleotides that are of alength that is too short to be capable of stably hybridizing to an SDImRNA under physiologic, in vivo conditions. Such an oligonucleotide maybe from about 6-10, or more nucleotides in length. To be used inaccordance with the present invention, such an oligonucleotide ispreferably modified to permit it to bind to a locus of the translationregion of an SDI-encoding mRNA. Examples of such modified moleculesinclude oligonucleotides bound to an antibody (or antibody fragment), orother ligand (such as a divalent crosslinking agent (such as, forexample, trimethylpsoralin, 8-methoxypsoralin, etc.) capable of bindingto a single-stranded SDI mRNA molecules.

An anti-SDI antisense oligonucleotide bound to one reactive group of adivalent crosslinking agent (such as psoralin (for example,trimethylpsoralin, or 8-methoxypsoralin) adduct would be capable ofcrosslinking to an SDI mRNA upon activation with 350-420 nm UV light.Thus, by regulating the intensity of such light (as by varying thewattage of the UV lamp, by increasing the distance between the cells andthe lamp, etc.) one may control the extent of binding between theantisense oligonucleotide and an SDI mRNA of a cell. This, in turn,permits one to control the degree of attenuation of SDI gene expressionin a recipient cell.

In general, the antisense oligomer is prepared in accordance with thenucleotide sequence of an SDI gene, and most preferably in accordancewith the nucleotide sequence of SDI-1 (FIGS. 5A-5D).

The sequence of the antisense oligonucleotide may contain one or moreinsertions, substitutions, or deletions of one or more nucleotidesprovided that the resulting oligonucleotide is capable of binding to orhybridizing with the above-described translation locus of either an SDImRNA, cDNA or an SDI gene itself.

Any means known in the art to synthesize the antisense oligonucleotidesof the present invention may be used (Zamechik et al., Proc. Natl. Acad.Sci. (U.S.A.) 83:4143 (1986); Goodchild et al., Proc. Natl. Acad, Sci.(U.S.A.) 85:5507 (1988); Wickstrom et al., Proc. Natl. Acad, Sci.(U.S.A.) 85:1028; Holt, J. T. et al., Mol. Cell. Biol. 8:963 (1988);Gerwirtz, A. M. et al., Science 242:1303 (1988); Anfossi, G., et al.,Proc. Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D., et al., EMBOJ. 8:3679 (1989); all of which references are incorporated herein byreference). Automated nucleic acid synthesizers may be employed for thispurpose. In addition, desired nucleotides of any sequence can beobtained from any commercial supplier of such custom molecules.

Most preferably, the antisense oligonucleotides of the present inventionmay be prepared using solid phase "phosphoramidite synthesis." Thesynthesis is performed with the growing nucleotide chain attached to asolid support derivatized with the nucleotide which will be the3'-hydroxyl end of the oligonucleotide. The method involves the cyclicalsynthesis of DNA using monomer units whose 5'-hydroxyl group is blocked(preferably with a 5'-DMT (dimethoxytrityl) group), and whose aminogroups are blocked with either a benzoyl group (for the amino groups ofcytosine and adenosine) or an isobutyryl group (to protect guanosine).Methods for producing such derivative are well known in the art.

The antisense and other inhibitor molecules of the present invention maybe used to immortalize valuable cell types (such as primary tissueculture cells, etc.) which would otherwise have a transient period ofproliferative viability. They may thus be used for research or to permitor facilitate the accumulation of large numbers of cells, as for organor tissue grafts or transplants. In one embodiment, therefore, theagents of the present invention may be used in conjunction with methodsfor organ or tissue culture to facilitate such methods.

A use is said to be therapeutic if it alters a physiologic condition. Anon-therapeutic use is one which alters the appearance of a user.

The agents of the present invention may be used topically orsystemically for a therapeutic or non-therapeutic purpose, such as, forexample, to counter the effects of aging, for example on skin tone,color, texture, etc., or on the degeneration of cells, tissue or organs,such as lymphocytes, vascular tissue (such as arteries, arterioles,capillaries, veins, etc.), liver, kidney, heart and other muscle, bone,spleen, etc. The agents of the present invention may be employed torejuvenate such cells, tissue or organs. Thus, they may be used inpharmaceuticals, and the like, which may comprise, for example, anantisense oligonucleotide, or its equivalent, and a lipophyllic carrieror adjunct, preferably dissolved in an appropriate solvent. Such asolvent may be, for example, a water-ethanol mixture (containing 10% to30% v/v or more ethanol. Such preparations may contain 000.1% to 1.0% ofthe antisense oligonucleotide. Suitable carriers, adjuncts an solventsare described in Remington's Pharmaceutical Science (16th ed., Osol, A.,Ed., Mack, Easton Pa. (1980), which reference is incorporated herein byreference).

Since the antisense and other inhibitor molecules of the presentinvention are capable of stimulating cellular proliferation, they may beused to promote wound healing recovery from burns, or after surgery, orto restore atrophied tissue, etc. For such an embodiment, these agentsmay be formulated with antibiotics, anti-fungal agents, or the like, fortopical or systemic administration.

Such antisense and other inhibitor molecules of the present inventionmay be used to stimulate the proliferation of spermatocytes, or thematuration of oocytes in humans or animals. Thus, the agents of thepresent invention may be used to increase the fertility of a recipient.

The molecules of the present invention may be used to provide genetherapy for recipient patients, In one embodiment, cells or tissue froma patient may be removed from the patient and treated with a molecule ofthe present invention under conditions sufficient to permit arestoration of an active growing state. In one preferred embodiment ofthis use, lymphocytes of an individual (such as, for example, an immunecompromised individual, such as an AIDS patient, etc., or animmune-competent individual who will serve as a donor of lymphocytes)can be removed and treated with antisense SDI nucleic acids. Theadministration of these molecules will derepress the lymphocytes. Afteradministration, the lymphocytes are reintroduced into the patient, andhave an enhanced ability to combat infection.

The molecules of the present invention are particularly suitable for usein the creation and/or study of animal models for disease or tissuedegeneration. Thus, the molecules of the present invention can be usedto study effectors of an animal model that is characterized by abnormalaging or cellular degeneration. Similarly, the administration of the SDImolecules (linked, for example to suitable regulatory sequences in orderto permit their expression in a recipient cell) can be used to createanimal models of aging and of tissue degeneration.

IV. Methods of Administration

The agents of the present invention can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thesematerials, or their functional derivatives, are combined in admixturewith a pharmaceutically acceptable carrier vehicle. Suitable vehiclesand their formulation, inclusive of other human proteins, e.g., humanserum albumin, are described, for example, in Remington's PharmaceuticalSciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980)). In order toform a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount of anantisense oligonucleotide, or its equivalent, or their functionalderivatives, together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Control release preparations may be achieved throughthe use of polymers to complex or absorb an antisense oligonucleotide,or its equivalent, or their functional derivatives. The controlleddelivery may be exercised by selecting appropriate macromolecules (forexample polyesters, polyamino acids, polyvinyl, pyrrolidone,ethylenevinylacetate, methylcellulose, carboxymethylcellulose, orprotamine, sulfate) and the concentration of macromolecules as well asthe methods of incorporation in order to control release. Anotherpossible method to control the duration of action by controlled releasepreparations is to incorporate an antisense oligonucleotide, or itsequivalent, or their functional derivatives, into particles of apolymeric material such as polyesters, polyamino acids, hydrogels,poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively,instead of incorporating these agents into polymeric particles, it ispossible to entrap these materials in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

The compositions of the present invention can also be formulated foradministration parenterally by injection, rapid infusion, nasopharyngealabsorption (intranasopharangeally), dermoabsorption, or orally. Thecompositions may alternatively be administered intramuscularly, orintravenously. Compositions for parenteral administration includesterile aqueous or nonaqueous solutions, suspensions, and emulsions.Examples of nonaqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers, adjuncts or occlusive dressings can beused to increase tissue permeability and enhance antigen absorption.Liquid dosage forms for oral administration may generally comprise aliposome solution containing the liquid dosage form. Suitable forms forsuspending liposomes include emulsions, suspensions, solutions, syrups,and elixirs containing inert diluents commonly used in the art, such aspurified water. Besides the inert diluents, such compositions can alsoinclude wetting agents, emulsifying and suspending agents, orsweetening, flavoring, coloring or perfuming agents.

A composition is said to be "pharmacologically acceptable" if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a "therapeutically effective amount" if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

Generally, the dosage needed to provide an effective amount of thecomposition will vary depending upon such factors as the recipient'sage, condition, sex, and extent of disease, if any, and other variableswhich can be adjusted by one of ordinary skill in the art.

Effective amounts of the compositions of the invention can vary from0.01-1,000 mg/ml per dose or application, although lesser or greateramounts can be used.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 CREATION OF THE cDNA LIBRARY

A cDNA library was obtained using RNA from normal human neonatalforeskin fibroblasts, such as the cell line HCA2. To do this, the cellswere grown in minimal essential medium with either Earle's or Hanks'balanced salt solution supplemented with 10% fetal bovine serum (GIBCOor Hyclone). Cells were cultured, and their in vitro life span wasdetermined, under the conditions disclosed by Smith, J. R., andBraunschweiger, K. I., J. Cell Physiol. 98:597-601 (1979), herebyincorporated by reference. Quiescent cells were made by replacing thenormal culture medium with culture medium containing 0.5% serum beforethe cells become confluent. The cells were maintained in low serumculture for up to 3 weeks.

Total cellular RNA was isolated either by the guanidium thiocyanate/CsClmethod (Garger, S. J. et al., Biochem. Biophys. Res. Commun. 117:835-842(1983)) or a guanidium thiocyanate/phenol method (Chomczynski, P., andSacchi, N., Anal. Biochem. 162:156-159 (1987), RNAzol B, Biotecx Lab.Inc. Texas). Poly A+ RNA was isolated by oligo (dT) cellulose columnchromatography (Collaborative Res. Massachusetts).

10 μg of the poly A+ RNA derived from senescent cells, as describedabove, was converted to double stranded cDNAs by using RNase H⁻ /MMLVreverse transcriptase according to the instructions of the supplier(BRL, MAD), and blunt-ended by T4 polymerase treatment. The doublestranded cDNA preparations were size fractionated by agarose gelelectrophoresis, and the 2-4.5 kb fraction isolated, for insertion intoan expression vector.

The expression vector used for this purpose was a 3.4 kb plasmid,designated pcDSRαΔ (FIG. 1). Plasmid pcDSRαΔ is a derivative of theplasmid pcDSRα296, which includes the Okayama-Berg SV40 promoter and theLTR from HTLV-1 (Takebe, Y. et al., Mol. Cell. Biol. 8:466-472 (1988);provided by Dr. M. Yoshida (Cancer Inst. of Japan)). Plasmid pcDSRαΔ wasformed by removing a 336 base pair (bp) segment of the Pstl-Kpnlfragment of pcDSRα296 and replacing it with 28 bp of a Pstl-Kpnlfragment from pUC19. The resulting plasmid (pcDSRαΔ) was used as acloning and expression vector.

Plasmid pSV2cat (Gorman, C. et al., Mol. Cell. Biol. 2:1044-1051 (1982))was provided by Dr. Gretchen Darlington (Texas Children's Hospital). ThepcD vector (Okayama, H., and Berg, P., Mol. Cell. Biol. 3:280-289(1983)) was provided by Dr. H. Okayama (Osaka University, Japan); theplasmid has the chloramphenicol acetyltransferase ("CAT") gene insertedbetween the SV40 promoter and SV40 poly A signal. pcDSRαΔ-cat wasconstructed from pcDSRαΔ by the insertion of 0.8 Kb of a HindIII-SmaIdigested SRα promoter fragment into HindIII digested pSVOcat via a twostep ligation. A very strong promoter was desired in order to allow forefficient expression screening of the cDNA library. From an analysis ofseveral mammalian expression vectors (pSV2cat, pcD-cat and pcDSRαΔ-cat,transfected into young cells), the SRα promoter was found to drive theexpression of the CAT gene at high efficiency in young cycling cells.The relative CAT activities of these plasmids were calculated bynormalizing to the amount of protein used for each reaction. Thetranscriptional efficiency was about 20-fold greater than that of theconventional pSV2 promoter, which utilizes the SV40 early gene promoter.

pCMVβ carries the E. coli β-galactosidase gene driven by the humancytomegalovirus immediate early gene promoter (MacGregor, G. R., andCaskey, C. T., Nucleic Acids Res. 17:2365 (1989); provided by Dr. GrantMacGregor, Baylor College of Medicine, Texas). Plasmid pβ440, whichcarries 443 bp of the human β-actin sequence (Nakajima-Iijima, S. etal., Proc. Natl. Acad. Sci. 82:6133-6137 (1985); provided by Dr. KozoMakino, Osaka University, Japan). Plasmid pHcGAP (Tso, J. Y. et al.,Nucleic Acids Res. 13:2485-2502 (1985)), which carries a full lengthhuman glyceraldehyde 3 phosphate dehydrogenase (GAPDH) cDNA, wasobtained from the American Type Culture Collection, Rockville, Md.

For cDNA antisense expression, full length cDNA fragments were excisedby BamHI digestion from the originally cloned pcDSRαΔ vector, andre-ligated in the reverse direction.

cDNAs recovered from the agarose gel were directly inserted into a calfintestine alkaline phosphatase treated SmaI site of pcDSRαΔ, andtransformed into E. coli MC1061 or DH-1. Ampicillin resistant colonieswere picked randomly and plasmid sizes determined. These procedures wererepeated until 2-4.5 kb cDNA insertions were achieved in more than 90percent of the plasmids tested. Then each E. coli colony was picked withtoothpicks and 5 colonies combined into one cDNA pool. More than 400cDNA pools were prepared, grown in 96 well microtiter plates and storedin 14% glycerol at -70° C. For DNA isolation, E. coli from each cDNApool was cultured in 200 ml, and treated by the standard methods ofethydium bromide/CsCl ultracentrifugation (Garger, S. J. et al.,Biochem. Biophys. Res. Commun. 117:835-842 (1983)) one or two times,followed by dialysis against TE (10 mM Tris pH 8.0, 1 mM EDTA) solution.

EXAMPLE 2 DEAE-DEXTRAN MEDIATED TRANSFECTION AND TRANSIENT EXPRESSIONSCREENING

Young, cycling fibroblast cells were seeded at a density of 0.9-1.2×10⁵per well in 6 well tissue culture plates or 35 mm tissue culture dishes18 h prior to transfection. Transfection was done as described byCullen, B. R., In: Guide to Molecular Cloning Techniques. Methods inEnzymology., S. L. Berger and A. R. Kimmel (ed.) Academic Press, pp.684-704 (1987); herein incorporated by reference with minormodifications as described below.

For each transfection, 100 ng of pCMVβ and 400 ng of a cDNA pool weremixed and suspended in 190 μl of phosphate buffered saline (PBS)solution and 10 μl of 10 mg/ml of DEAE-dextran (Pharmacia, MW ⁻ 500,000)was added. 400 ng of the cloning vector plasmid, pcDSRαΔ, was used withpCMVβ as a control. After washing the cells with PBS once, DNA solutionswere added and the cells incubated for up to 45 min at 37° C. in a CO₂incubator. Then 2 ml of cell culture medium with serum, containing 64 μMchloroquine (Sigma, MO) was added directly and incubated for another 2.5h. After the chloroquine treatment, the transfection mixture was removedand the cells treated with 10% dimethyl sulfoxide in cell culture mediumwith serum for 2 min. Cells were then returned to fresh cell culturemedium with serum and incubated to allow for expression of thetransfected DNA.

18 h after transfection, 0.5 μCi/ml of 3H-thymidine was added and theincubation continued for another 48 h. Cells were fixed by adding 25 μlof 25% of glutaraldehyde solution to the culture medium and incubatedfor 5 min at room temperature, followed by three washings with PBS.Immediately after washing, cells were treated with the X-gal reactionmixture (1 mM MgCl₂, 3 mM K₄ [Fe(CN)₆ ], 3 mM K₃ [Fe(CN)₆ ], 0.1% tritonX-100, and 1 mM X-gal dissolved in 0.1M sodium phosphate buffer (pH 7.5)containing 10 mM KC1) for up to 20 min to allow light-blue staining ofthe cells. After the X-gal staining, the cells were washed with water,dried and processed for autoradiography using Kodak NTB nuclear trackemulsion (Kodak, NY). DNA synthesis activity in X-gal positive cells wasthen determined. The percent inhibition of DNA synthesis was calculatedusing the formula: ##EQU1##

Candidate cDNA pools were divided into individual cDNAs and screenedfurther for the identification of specific DNA synthesis inhibitory cDNAsequences.

Nuclear microinjection of young cycling cells was performed as describedby (Lumpkin, C. K. et al., Mol. Cell Biol. 6:2990-2993 (1986), hereinincorporated by reference). Briefly, 5,000-10,000 cells were plated onto22 mm square etched grid coverslips (Bellco) in 35 mm tissue culturedishes. Three or four days later, nuclear microinjections were performedon a minimum of 300 cells, using either pCMVβ+cDNA plasmid orpCMVβ+pcDSRαΔ (which served as the control). Plasmids wereco-microinjected at a concentration of 50 ng/μl each. 18 hours aftermicroinjection, the cells were labeled with ³ H-thymidine for 24 h,fixed, stained with X-gal and processed for autoradiography. The percentinhibition of DNA synthesis was calculated as above.

Northern blot analysis was performed using either 5 μg of total RNA or 1μg poly A+ RNA. The RNA was size fractionated by electrophoresis onformaldehyde-agarose gels and transferred to nylon membranes (ICN;Biotrans, formerly Pall Biodyne A) as described by Maniatis, T. et al.,Molecular cloning: A Laboratory Manual; Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982), herein incorporated by reference.Radioactive probes were prepared by the random primer method, and blotshybridized as described by Maniatis, T. et al., Molecular cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

The northern blot analyses revealed that the sizes of the cellulartranscripts of the SDIs were compatible with the sizes of the SDI cDNAs.This was expected since successful expression screening requiresfull-length cDNA insertions into the vector.

For rehybridization with β-actin or glyceraldehyde phosphatedehydrogenase (GAPDH) probe, filters were repeatedly stripped of thelabelled probes following the manufacturer's instructions. The data werequantitated by an Ambis Radioanalytic Scanning System.

An assay of CAT activity was determined as follows: Young cycling cellswere seeded into 35 mm dishes and 500 ng of plasmid transfected asdescribed above. 24h after the transfection, the cells were scraped fromthe dish, and CAT assay performed as described by Gorman (Gorman, C.,In: DNA Cloning, A Practical Approach. IRL Press, Oxford, England, pp.143-164 (1985), herein incorporated by reference).

EXAMPLE 3 CDNA CLONING OF THE SENESCENT CELL DERIVED INHIBITORS (SDI) OFDNA SYNTHESIS

Double stranded cDNAs were synthesized from senescent cell derived polyA+ RNA, which has been shown to inhibit DNA synthesis in young cellswhen microinjected into the cytoplasm (Lumpkin, C. K. et al., Science232:393-395 (1986)). The cDNAs were size fractionated, inserted intopcDSRαΔ. The resulting E. coli clones were divided into small pools.Plasmids from each pool were co-transfected with the transfection markerplasmid, pCMVβ, which allowed a determination of the labelling index oftransfected cells specifically, since even in high efficiencytransfection, frequencies varied from experiment to experiment.Transfection frequencies of the marker plasmid ranged from 30-90%. About200 cDNA pools were screened and four pools remained positive for DNAsynthesis inhibitory activity after five repeated transfections. Thecandidate pools were then divided into individual plasmids and screenedfurther.

Three independent positive plasmid clones were obtained. In the cDNApool A, only one plasmid, No. 2, exhibited strong DNA synthesisinhibitory activity. Similarly, in pools B and C only one cDNA clonecaused inhibition. The size of inserted cDNAs was 2.1 kb, 1.2 kb and 2.7kb, respectively. These cDNA sequences have been designated as senescentcell derived inhibitors, SDI-1, SDI-2 and SDI-3, respectively.

The nucleotide sequence of the SDI-1 cDNA clone (SEQ ID NO: 1), and theamino acid sequence of SDI-1 (SEQ ID NO: 2) have been determined. ThecDNA sequence presented herein for SDI-1 differs from that described inU.S. patent application Ser. No. 07/808,523 in possessing an unrecited Gat position 286, and in having the sequence CG rather than GC atposition 1843-1844. The presently disclosed sequence was obtainedthrough the resequencing of the pcDSRαΔ-SDI-1 plasmid whose isolationand characteristics were described in U.S. patent application Ser. No.07/808,523. E. coli DH5 transformed with the pcDSRαΔ-SDI-1 plasmid wasdeposited with the American Type Culture Collection, Rockville, Md.,U.S.A., on Oct. 1, 1992, and has been accorded accession number ATCC69081.

EXAMPLE 4 MICROINJECTION OF SDI SEQUENCES INTO YOUNG CYCLING CELLS

In order to verify the functional activity of SDI sequences,microinjections were performed. A plasmid carrying either SDI-1 or SDI-2was co-microinjected with the marker plasmid into the nuclei of youngcycling cells. The labelling index of the resulting blue cells wasdetermined (Table 1). These plasmids showed strong inhibitory activityon DNA synthesis of young cells. For control experiments, the emptyvector was co-microinjected with the marker plasmid. This caused slightinhibition when the labelling index was compared with uninjected cells,a phenomenon also observed in transfection experiments. Microinjectionswith SDI-3 were not performed because the inhibitory activity was lowerthan SD-I and SD-2 transfection experiments.

                                      TABLE 1                                     __________________________________________________________________________    Microinjection of Various Plasmids Into Young Cycling Cells                                     No. of Labelled                                             Plasmids   No. of Cells                                                                         Nuclei     Labelling                                                                           %                                          Injected   Injected                                                                             Per Total Blue Cells*                                                                    Index (%)                                                                           Inhibition                                 __________________________________________________________________________    Exp. 1                                                                            pCMVβ +                                                                         335    58/97      59.8  0                                              pcDSRαΔ                                                           pCMVβ +                                                                         380    20/89      22.5  62.4                                           SDI-1                                                                         pCMVβ +                                                                         380     6/82      7.3   87.8                                           SDI-2                                                                     Exp. 2                                                                            pCMVβ +                                                                         423     68/109    62.3  0                                              pcDSRαΔ                                                           pCMVβ +                                                                         465    26/98      26.5  57.5                                           SDI-1                                                                         pCMVβ +                                                                         475     27/118    22.9  63.2                                           SDI-2                                                                     __________________________________________________________________________       Control                                                                     *This is the number of cells expressing detectable levels of                  galactosidase.                                                                The concentration of each DNA was 50 μg/ml.                           

EXAMPLE 5 ANTISENSE DNA TRANSFECTION

In order to examine whether any inhibitory activities are sequenceorientation specific, antisense expression vectors of SDI-1 and SDI-2sequences were constructed. Since both sequences lacked BamHI sites andsince BamHI sites were present at both ends of the cDNA (FIG. 1), thesequences were easily excised and religated in the opposite orientation.Transfection of antisense sequences resulted in no inhibition of DNAsynthesis in young cells (FIG. 3). In addition, no enhancement wasobserved. The results clearly indicate the sequence orientationspecificity of the SDI activity, and suggest the presence of specificgene products coded by the cDNA sequences.

EXAMPLE 6 EXPRESSION OF SDI mRNAS DURING CELLULAR SENESCENCE

To examine the changes in SDImRNA expression during cellular senescence,total RNA from young and senescent cells was hybridized to 32P-labelledSDt cDNA probes. The SDI-1 probe hybridized to a 2.1 kb cellulartranscript, SDI-2 hybridized to a 1.4 kb transcript, and SDI-3hybridized to a 2.5 kb transcript (Table 2). Table 2 provides aquantitation of the total RNA northern analysis of expression of SDIgenes in young (Y) and senescent (S) cells. 5 μg each of total RNA fromyoung and senescent cells were hybridized with SDI probes. The filterswere repeatedly stripped of the radioactive probe and rehybridized withthe probes for the internal controls. The relative amount of SDI mRNA ineach sample was normalized by the amount of GAPDH detected on the samefilter and by the relative amount of SDI/GAPDH.

                  TABLE 2                                                         ______________________________________                                        Quantitation of the Northern Analysis                                                         SDI-1  SDI-2    SDI-3                                         ATTRIBUTE         Y     S      Y   S    Y   S                                 ______________________________________                                        Relative Amount of SDI                                                                          1.0   3.3    1.0 0.31 1.0 0.31                              Relative Amount of GAPDH                                                                        1.0   0.37   1.0 0.36 1.0 0.38                              Relative Amount of                                                                              1.0   9.3    1.0 0.86 1.0 0.82                              SDI/GAPDH                                                                     ______________________________________                                    

During cellular senescence, the SDI-1 message increased about 3-fold,while SDI-2 and SDI-3 messages decreased 3-fold. The same filters wererehybridized with a β-actin, and then to a GAPDH probe as internalcontrols. The results demonstrated that expression of both control genesdecreased about 3-fold during cellular senescence. In previous studies,a 2-3 fold decrease of β-actin expression during cellular senescence hadbeen observed (Kumazaki, T. et al., Exp. Cell Res. 195:13-19 (1991);Seshadri, T., and Campisi, J., Science 247:205-209 (1990); Furth, J. J.,J. Gerontol. 46:B122-124 (1991)). The decreased expression of bothβ-actin and GAPDH genes in senescent cells led to the use of poly A+ RNAfor northern analysis. Poly A+ RNA was isolated from the total cellularRNA preparations used for Table 2, and hybridized to SDI cDNA, followedby probing with β-actin and GAPDH respectively (Table 3). Table 3discloses the results of a poly A+ RNA Northern analysis of SDI geneexpression in young (Y) and senescent (S) cells. 1 μg each of poly A+RNA from young and senescent cells were used for the analyses. Therelative amount of SDI mRNA in each sample was calculated as in Table 2.

                  TABLE 3                                                         ______________________________________                                        Quantitation of the Northern Analysis                                                         SDI-1  SDI-2    SDI-3                                         ATTRIBUTE         Y     S      Y   S    Y   S                                 ______________________________________                                        Relative Amount of GAPDH                                                                        1.0   0.83   1.0 0.87 1.0 0.87                              Relative Amount of                                                                              1.0   11.4   1.0 1.0  1.0 1.0                               SDI/GAPDH                                                                     ______________________________________                                    

The results clearly indicated that the expression of both β-actin andGAPDH was equal in young and senescent cells when they were compared onthe basis of mRNA, consistent with previous observations. When SDI geneexpression was compared at the mRNA level, SDI-1 mRNA was increased11-fold in senescent cells, whereas expression of SDI-2 and SDI-3remained constant throughout the in vitro lifespan (Table 3). Thisresult suggests that SDI-1 is a senescent cell specific inhibitor of DNAsynthesis, whereas SDI-2 and SDI-3 are most likely more generalinhibitors involved in cell cycle regulation.

EXAMPLE 7 CHANGES OF POLY A RNA CONTENT DURING CELLULAR SENESCENCE

The observation that the results of the total versus poly A+ RNAnorthern analyses were quantitatively different, indicated that the polyA+ RNA content in total RNA preparations might change during cellularsenescence. To test this hypothesis, cells were cultivated serially andtotal RNA was harvested at different population doubling levels. Poly A+RNA was isolated from each sample.

The result clearly indicated that poly A+ RNA content decreasedgradually during cellular senescence (FIG. 4). In FIG. 4, cells werecultivated serially and total RNA was harvested. Poly A+ RNA: % of totalRNA was plotted against the culture's age (% in vitro life spancompleted). Senescent cells had 3-4 fold less poly A+ RNA when comparedwith very young cells. However, when total RNA content per cell wascalculated, senescent cells had 1.3-1.5 fold more than young cells (see,Cristofalo, V. J., and Kritchevsky, D., Med. Exp. 19:313-320 (1969)).

In order to determine whether SDI-1 message increased gradually duringsubcultivation or whether a rapid increase occurred near the end of thein vitro life span, poly A+ RNA from cultures at different populationdoublings was hybridized with the ³² p labelled SDI-1 probe. Thisanalysis revealed that SDI-1 expression increased as the cultures becamesenescent, with a major change occurring during the final few passages(Table 4). Table 4 shows the accumulation of SDI-1 mRNA during cellularaging process. 1 μg each of poly A+ RNA from the cells of differentpopulation doublings were hybridized to SDI-1 probe. The relative amountof SDI-1 mRNA in each sample was calculated as in Table 2.

                  TABLE 4                                                         ______________________________________                                        Quantitation of % Lifespan Completed                                          ATTRIBUTE  24%    37%    46%  66%  78%  88%  100%                             ______________________________________                                        Relative Amount                                                                          1.0    1.6    1.5  1.3  1.4  1.3  0.9                              of GAPDH                                                                      Relative Amount                                                                          1.0    2.2    2.1  4.0  3.5  6.2  20.5                             of SDI/GAPDH                                                                  ______________________________________                                    

Changes in SDI-1 expression during quiescence were also examined. Young,quiescent cells were maintained in 0.5% fetal bovine serum(FBS)-containing medium for up to three weeks. Total RNA was harvestedeach week and the amount of RNA hybridizing to the SDI-1 probe wasanalyzed. SDI-1 message increased significantly during cellularquiescence (Table 5). Table 5 shows the accumulation of SDI-1 mRNAduring cellular quiescence. 4 μg each of total RNA was obtained from theyoung cells cultured with 0.5% FBS containing medium for 1, 2, 3 weeks,was hybridized with SDI-1 probe. The relative amount of SDI-1 mRNA wascalculated as in Table 2 (C: control culture with 10% FBS medium). Whenthe result was normalized to GAPDH expression, SDI-1 expression wasfound to have increased 18-fold after two weeks in low serum mediumcompared to that of a control dividing culture in 10% FBS medium.

                  TABLE 5                                                         ______________________________________                                        Accumulation of SDI-1 mRNA During                                             Cellular Quiescence                                                           ATTRIBUTE         C     1 wk     2 wk 3 wk                                    ______________________________________                                        Relative Amount of GAPDH                                                                        1.0   0.72     0.88 0.37                                    Relative Amount of                                                                              1.0   12.2     18.4 14.9                                    SDI/GAPDH                                                                     ______________________________________                                    

The fact that the cellular representation of mRNA vs total RNA was foundto change during cellular senescence is significant. During the in vitroaging process, the content of mRNA was found to decrease gradually (FIG.4), in spite of the slight increase of the total RNA per cell. Thisphenomenon indicates that a gradual decline of the overall geneexpressions during the cellular aging process, and explains thedecreased expression of β-actin and GAPDH genes in senescent cells whenNorthern blot analysis was done with total RNA (Table 2). However, theexpression levels of these housekeeping genes between young andsenescent cells were almost constant when the Northern blot analysis wasdone with poly A+ RNAs (Table 3). This analysis revealed the strongexpression of SDI-1 message in senescent cells, and unchangingexpression of SDI-2 and 3 genes throughout the in vitro life span.

EXAMPLE 8 THE SDI-1 GENE

The SDI-1 gene codes for a senescent cell specific inhibitor of DNAsynthesis. Increased expression of this gene occurred when the cellsentered their final few divisions (Table 4). The expression kineticscorrelated well with the phenotypic expression of senescence cells.SDI-1 gene expression was also found to increase after young cells weremade quiescent and nondividing by serum deprivation (Table 5). Thisresult demonstrates the involvement of this gene in the inhibition ofDNA synthesis of cellular quiescence as well as senescence. Cells madequiescent by deprivation of serum growth factors have been shown toproduce an inhibitor of DNA synthesis with characteristics similar tothe inhibitor from senescent cells (Pereira-Smith, O. M. et al., Exp.Cell Res. 160:297-306 (1985); Stein, G. H., and Atkins, L., Proc. Natl.Acad. Sci. USA.83:9030-9034 (1986)).

The fact that SDI-1 expression increases during both senescence andquiescence indicates that it is an inhibitor of DNA synthesis (Smith, J.R., J. Gerontol. 45:B32-35 (1990); herein incorporated by reference).Alternatively, SDI-1 sequences might be related to the growtharrest-specific genes recently cloned from mouse cells (Schneider, C. etal., Cell 54:787-793 (1988); Manfioletti, G. et al., Mol. Cell. Biol.10:2924-2930 (1990)).

EXAMPLE 9 THE EXPRESSION OF THE SDI-1 GENE PRODUCT

SDI-1 cDNA has been expressed in two different bacterial expressionsystems, has been transcribed in vitro and translated in two differentin vitro systems. Two bacterial expression systems were used in order tomaximize the probability of obtaining sufficient amounts of SDI-1protein. In the first expression system, SDI-1 protein was expressed asa glutathione S-transferase fusion protein at yields of 5-10 μg perliter of bacterial culture. The recombinant protein could be cleavedwith thrombin and purified in order to give an SDI-1 protein with a fewextra amino acids. In the second expression system, a 6 histidine aminoterminal tag was utilized in order to aid in purification. Thisrecombinant protein may be used without further modification. Bothsystems permitted the isolation of pure preparations of protein.

In the course of this experiment, in vitro transcription and translationsystems were used to confirm the open reading frame deduced from thenucleic acid sequence of the SDI-1 cDNA. The calculated molecular weightof the SDI-1 protein is approximately 16,000 daltons. The in vitrosynthesized protein migrates, by SDS PAGE, with a relative mobility ofapproximately 21,000 daltons. This small difference may be due to aslightly unusual charge or conformation of the SDI-1 protein. A partialamino acid sequence of the bacterially expressed protein verified theopen reading frame (SEQ ID NO:2).

The bacterially expressed proteins were used to generate polyclonalantisera and monoclonal antibodies to the intact native protein. Suchantibodies may be more effective in immunoprecipitation of SDI-1 proteinand SDI-1 protein complexes than the antisera produced from syntheticpeptides. Preliminary immunocytochemical studies, using an antisera ofhighest affinity (antisera #55) which reacted strongly with the fusionprotein on a western transfer at a 1:20,000 dilution, suggested that theSDI-1 protein was relatively abundant in senescent cells compared todividing young cells. In senescent cells the location appears to beperinuclear, whereas in young cells there appears to be a small amountof SDI-1 protein located in the nucleus. In order to obtain specificstaining it was necessary to pre-absorb the antisera against a fixedcell monolayer of cells which do not express detectable levels of SDI-1mRNA (TE85). The cells were fixed with 4% paraformaldehyde followed bymethanol.

In order to study the cellular phenotype resulting from the inducedexpression of SDI-1 mRNA in cells which normally express the gene at lowlevels and to examine the effect of antisense SDI-1 constructs it isdesirable to obtain cell lines in which the SDI-1 gene is stablyintegrated under the control of an inducible promoter. Toward this goal,a functional vector containing SDI-1 under the control of themetallothionine promoter was constructed. Following transfection of thisconstruct into young proliferation competent cells and incubation in thepresence of 100 μM zinc chloride and 2 μM cadmium chloride, initiationof DNA synthesis was inhibited by about 50%. In the absence of metalsthere was no inhibition of DNA synthesis. The inhibitory activityobserved is not due to metal toxicity since cells transfected with thecontrol vector (pcDSRα) and grown in the presence of metals were foundto have approximately 90% of the DNA synthetic capacity of cellstransfected with the same plasmid grown in the absence of metals.

In order to demonstrate that the inhibitory effects observed with SDI-1were not related to the nature of the specific promoter used to driveexpression, the capacity of SDI-1, expressed from other promoters, toinhibit DNA synthesis was investigated. Young proliferating humanfibroblasts were therefore co-transfected with CMV-β-gal and CMV-SDI-1.Transfection of cells with CMV-β-gal had little effect on DNA synthesiswhile CMV-SDI-1 was even more effective than SDI-1 in the pcDSRα vectorin these particular experiments.

The SV40 large T antigen is capable of inducing senescent cells tosynthesize DNA. It was therefore of interest to determine whether theinhibitory action of SDI-1 could be overcome by the expression of Tantigen. Moreover, it was desirable to determine that the action ofSDI-1 was not due to the induction of a general metabolic imbalance incells. If such were the case, one would not expect large T antigen toantagonize its effect. For these reasons, cells were co-transfected withSDI-1 cDNA and vectors in which T antigen was driven by the CMVpromoter. Such co-transfection experiments revealed that the inhibitoryactivity of SDI-1 was largely abolished by the co-expression of the SV40large T antigen.

Transient transfection assays were performed using an additional normalhuman fibroblast cell line (neonatal foreskin cell line (CSC303) and theWI38 immortal cell line in order to determine the generality of theinhibitory effect of SDI-1. In both cases, significant inhibition(40-50%) was observed. Furthermore, SDI-1 was found to inhibit SUSMI(40%) but not an SV40 transformed cell line GM639 or HeLa cells (<20%).The results thus far are consistent with earlier results obtained fromheterokaryon experiments in which HeLa cells and cells transformed withSV40 virus were not inhibited by fusion with senescent cells. Thisprovides further evidence that SDI-1 behaves like the inhibitorpreviously detected in senescent cells.

EXAMPLE 10 SOUTHERN ANANLYSIS OF THE SDI-1 GENE

In order to determine whether the absence or inactivity of SDI-1 wasresponsible for cellular immortality in any of the four complementationgroups for indefinite division, genomic DNA and mRNA was examined fromcell lines representative of the four groups. Southern analysis revealedthe expected 5 and 10 kb bands after digestion with Eco RI. Therefore,no gross deletions or rearrangements have occurred in the SDI-1 gene inthese cell lines. By Northern analysis, it was determined that SDI-1mRNA was lower or absent in the cell lines that had been assigned tocomplementation groups B and C. SDI-1 was present at higher levels incell lines representative of complementation groups A and D. Thisresults suggests that part of the mechanism by which the cell lines mayhave escaped cellular senescence is through the loss of ability toexpress sufficient levels of the active SDI-1 gene.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2106 base pairs                                                   (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI- SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Homo sapiens                                                    (G) CELL TYPE: SENESCENT HUMAN CELLS                                           (vii) IMMEDIATE SOURCE:                                                      (A) LIBRARY: SENESCENT CELL DERIVED CDNA LIBRARY                              (B) CLONE: SDI-1                                                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCTGCCGAAGTCAGTTCCTTGTGGAGCCGGAGCTGGGCGCGGATTCGCCGAGGCACCGAG60                GCACTCAGAGGAGGCGCCATG TCAGAACCGGCTGGGGATGTCCGTCAGAACCCATGCGGC120              AGCAAGGCCTGCCCCCGCCTCTTCGGCCCAGTGGACAGCGAGCAGCTGAGCCGCGACTGT180               GATGCGCTAATGGCGGGCTGCATCCAGGAGGCCCGTGAGCGATGGAACTTCGACTTTGTC240               A CCCAGACACCACTGGAGGGTGACTTCGCCTGGGAGCGTGTGCGGGGCCTTGGCCTGCCC300              AAGCTCTACCTTCCCACGGGGCCCCGGCGAGGCCGGGATGAGTTGGGAGGAGGCAGGCGG360               CCTGGCACCTCACCTGCTCTGCTGCAGGGGACAGCAGAGGAAGACCATG TGGACCTGTCA420              CTGTCTTGTACCCTTGTGCCTCGCTCAGGGGAGCAGGCTGAAGGGTCCCCAGGTGGACCT480               GGAGACTCTCAGGGTCGAAAACGGCGGCAGACCAGCATGACAGATTTCTACCACTCCAAA540               CGCCGGCTGATCTTCTCCAAGAGGAAGCC CTAATCCGCCCACAGGAAGCCTGCAGTCCTC600              GAAGCGCGAGGGCCTCAAAGGCCCGCTCTACATCTTCTGCCTTAGTCTCAGTTTGTGTGT660               CTTAATTATTATTTGTGTTTTAATTTAAACACCTCCTCATGTACATACCCTGGCCGCCCC720               CTGCCCCCC AGCCTCTGGCATTAGAATTATTTAAACAAAAACTAGGCGGTTGAATGAGAG780              GTTCCTAAGAGTGCTGGGCATTTTTATTTTATGAAATACTATTTAAAGCCTCCTCATCCC840               GTGTTCTCCTTTTCCTCTCTCCCGGAGGTTGGGTGGGCCGGCTTCATGCCAGCTAC TTCC900              TCCTCCCCACTTGTCCGCTGGGTGGTACCCTCTGGAGGGGTGTGGCTCCTTCCCATCGCT960               GTCACAGGCGGTTATGAAATTCACCCCCTTTCCTGGACACTCAGACCTGAATTCTTTTTC1020              ATTTGAGAAGTAAACAGATGGCACTTTGARGGGG CCTCACCGAGTGGGGGCATCATCAAA1080             AACTTTGGAGTCCCCTCACCTCCTCTAAGGTTGGGCAGGGTGACCCTGAAGTGAGCACAG1140              CCTAGGGCTGAGCTGGGGACCTGGTACCCTCCTGGCTCTTGATACCCCCCTCTGTCTTGT1200              GAAGGCAGGGG GAAGGTGGGGTCCTGGAGCAGACCACCCCGCCTGCCCTCATGGCCCCTC1260             TGACCTGCACTGGGGAGCCCGTCTCAGTGTTGAGCCTTTTCCCTCTTTGGCTCCCCTGTA1320              CCTTTTGAGGAGCCCCAGCTACCCTTCTTCTCCAGCTGGGCTCTGCAATTCCCCTCT GCT1380             GCTGTCCCTCCCCCTTGTCCTTTCCCTTCAGTACCCTCTCAGCTCCAGGTGGCTCTGAGG1440              TGCCTGTCCCACCCCCACCCCCAGCTCAATGGACTGGAAGGGGAAGGGACACACAAGAAG1500              AAGGGCACCCTAGTTCTACCTCAGGCAGCTCAAG CAGCGACCCCCCCCTCCTCTAGCTGTl560             GGGGGTGAGGGTCCCATGTGGTGGCACAGGCCCCCTTGAGTGGGGTTATCTCTGTGTTAG1620              GGGTATATGATGGGGGAGTAGATCTTTCTAGGAGGGAGACACTGGCCCCTCAAATCGTCC1680              AGCGACCTTCC TCATCCACCCCATCCCTCCCCAGTTCATTGCACTTTGATTAGCAGCGGA1740             ACAAGGAGTCAGACATTTTAAGATGGTGGCAGTAGAGGCTATGGACAGGGCATGCCACGT1800              GGGCTCATATGGGGCTGGGAGTAGTTGTCTTTCCTGGCACTAAGCTTGAGCCCCTG GAGG1860             CACTGAAGTGCTTAGTGTACTTGGAGTATTGGGGTCTCACCCCAAACACCITCCAGCTCC1920              TGTAACATACTGGCCTGGACTGTTTTCTCTCGGCTCCCCATGTGTCCTGGTTCCCGTTTC1980              TCCACCTAGACTGTAAACCTCTCGAGGGCAGGG ACCACACCCTGTACTGTTCTGTGTCTT2040             TCACAGCTCCTCCCACAATGCTGATATACAGCAGGTGCTCAATAAACGATTCTTAGTGAA2100              AAAAAA2106                                                                    (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 164 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI- SENSE: NO                                                          (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: HOMO SAPIENS                                                   (B) STRAIN: SDI-1                                                             (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Senescent cell derived cDNA library                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerGluProAlaGlyAspValArgGlnAsnProCysGlySerLys                              15 1015                                                                       AlaCysArgArgLeuPheGlyProValAspSerGluGlnLeuSerArg                              202530                                                                        AspCysAspAlaLeuMetAlaGlyCysIl eGlnGluAlaArgGluArg                             354045                                                                        TrpAsnPheAspPheValThrGluThrProLeuGluGlyAspPheAla                              505560                                                                        TrpGl uArgValArgGlyLeuGlyLeuProLysLeuTyrLeuProThr                             65707580                                                                      GlyProArgArgGlyArgAspGluLeuGlyGlyGlyArgArgProGly                               859095                                                                       ThrSerProAlaLeuLeuGlnGlyThrAlaGluGluAspHisValAsp                              100105110                                                                     LeuSerLeuSerCysThrLeu ValProArgSerGlyGluGlnAlaGlu                             115120125                                                                     GlySerProGlyGlyProGlyAspSerGlnGlyArgLysArgArgGln                              130135 140                                                                    ThrSerMetThrAspPheTyrHisSerLysArgArgLeuIlePheSer                              145150155160                                                                  LysArgLysPro                                                              

What is claimed is:
 1. A purified protein, being substantially free ofnatural contaminants and capable of inhibiting DNA synthesis in arecipient cell, wherein said protein is senescent cell derivedinhibitor-1, SDI-1 comprising the amino acid sequence of SEQ ID NO:2.